Monocyte unresponsiveness and impaired IL1β, TNFα and IL7 production are associated with a poor outcome in Malawian adults with pulmonary tuberculosis

Abstract

Background: Early death during TB treatment is associated with depressed TNFα response to antigenic stimulation and propensity to superadded bacterial infection. Hypothesising the role of monocyte unresponsiveness, we further compared the immunological profile between patients who died or suffered a life-threatening deterioration ('poor outcome') during the intensive phase of TB treatment with patients who had an uneventful clinical course ('good outcome') who had been recruited as part of a larger prospective cohort study of Malawian TB patients.

Methods: Using Luminex, IL1β, IL2, IL4, IL5, IL6, IL7, IL8, IL10, IL12, IL13, IL17, GCSF, GMCSF, MCP1, MIP1b, IFNγ and TNFα were measured in whole blood assay supernatants (stimulated with Mycobacterium tuberculosis H37Rv and LPS) and serum from 44 Malawian adult TB patients (22 of each outcome) immediately prior to commencing treatment, after 7 days and on day 56 of TB treatment. Monocyte surface expression of CD14, CD16, TLR2, TLR4, CD86 and HLADR, and intracellular TNFα were measured by flow cytometry as was intracellular TNFα response to purified TLR ligands.

Results: Lower TB antigen-induced IL1β (p = 0.006), TNFα (p = 0.02) and IL7 (p = 0.009) were produced in the poor outcome group. TNFα was produced by 'classical' CD14(hi)CD16(lo) monocytes, with no correlation between this response and expression of monocyte surface markers. Response to TB antigens correlated with responses to the purified TLR 2, 3 and 4 ligands.

Conclusions: Dysregulated monocyte cytokine production was identified in TB patients with poor outcome. Lower TNFα responses to H37Rv paralleled lower responses to a panel of TLR ligands, suggesting an underlying perturbation in common TLR signalling pathways. Future work should explore the role of TLR polymorphisms in immune response and clinical outcome in TB patients.

Fig. 1

Flowchart indicating the relationship of i) the case–control study and ii) the flow cytometry and ICS study in relationship to the parent study (Waitt et al., JID 2011)

Fig. 2

Cytokine responses to stimulation with H37Rv and LPS, expressed as the area under the concentration time curve (AUC) between Day 0 (immediately prior to commencing treatment) and Day 7 of TB treatment. Patients who had a poor outcome had lower production of TNFα, IL1β and IL7 compared to matched control patients who had an uneventful clinical course. No significant differences were identified between patient groups in the other cytokines and chemokines analysed

Fig. 3

IL1β and TNFα production in response to stimulation with H37Rv and LPS immediately prior to the start of TB treatment and at the end of the intensive phase, in 6 patients who had a life-threatening clinical deterioration (poor) with controls matched by age, sex, HIV status and CD4 count who had an uneventful clinical course. There was significant restoration of the depressed IL1β responses in parallel with clinical recovery (p = 0.04), with a trend towards restoration of TNFα response (0.18)

Fig. 4

TNFα production in response to stimulation with H37Rv and LPS in ‘classical’ CD14^hiCD16^lo and CD14^loCD16^hi monocyte populations. A significantly greater percentage of CD14^hiCD16^lo cells produced TNFα in response to stimulation with H37Rv (p = 0.0002) and LPS (p < 0.0001)